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Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion [GSIS]. However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor [GEF] for Rac1, in GSIS in pancreatic β-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation [i.e., GTP-bound form] and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.

DS. Heart failure attenuates muscle metaboreflex control of ventricular contractility during dynamic exercise. Am J Physiol Heart Circ Physiol 292: H2159 -H2166, 2007. First published December 22, 2006; doi:10.1152/ajpheart.01240.2006.-Underperfusion of active skeletal muscle elicits a reflex pressor response termed the muscle metaboreflex (MMR). In normal dogs during mild exercise, MMR activation causes large increases in cardiac output (CO) and mean arterial pressure (MAP); however, in heart failure (HF) although MAP increases, the rise in CO is virtually abolished, which may be due to an impaired ability to increase left ventricular contractility (LVC). The objective of the present study was to determine whether the increases in LVC seen with MMR activation during dynamic exercise in normal animals are abolished in HF. Conscious dogs were chronically instrumented to measure CO, MAP, and left ventricular (LV) pressure and volume. LVC was calculated from pressure-volume loop analysis [LV maximal elastance (E max) and preload-recruitable stroke work (PRSW)] at rest and during mild and moderate exercise under free-flow conditions and with MMR activation (via partial occlusion of hindlimb blood flow) before and after rapid ventricular pacing-induced HF. In control experiments, MMR activation at both workloads [mild exercise (3.2 km/h) and moderate exercise (6.4 km/h at 10% grade)] significantly increased CO, E max, and PRSW. In contrast, after HF was induced, CO, Emax, and PRSW were significantly lower at rest. Although CO increased significantly from rest to exercise, E max and PRSW did not change. In addition, MMR activation caused no significant change in CO, E max, or PRSW at either workload. We conclude that MMR causes large increases in LVC in normal animals but that this ability is abolished in HF. pressor response; elastance; preload recruitable stroke work WHEN EXERCISING SKELETAL MUSCLE does not receive sufficient blood flow to meet the ongoing metabolic demands, by-products of metabolism such as lactic acid, H ϩ , adenosine, potassium, diprotonated phosphate, and arachidonic acid products, among others, accumulate within the muscle and stimulate group III and IV afferent neurons, which evokes a reflex response known as the muscle metaboreflex (MMR). This reflex response consists of increases in efferent sympathetic nerve activity (SNA) and mean arterial pressure (MAP) (1, 3, 11, 17, 18, 24, 31-35, 39, 40, 43, 46, 47, 52, 53). In addition, MMR activation causes increases in cardiac output (CO), heart rate (HR), and plasma levels of vasoactive hormones and produces vasoconstriction in the renal and the nonischemic active skeletal muscle vasculature to partially restore arterial O 2 delivery and blood flow to the hypoperfused muscles (25,33,36). The rise in CO likely results from increases in ventricular performance, HR, and central blood volume mobilization (32,42,43). By this means the MMR-induced increases in ventricular performance act to slightly increase or sustain stroke volume (SV) despite decreas...

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C2-ceramide, a cell permeable analogue of ceramide [CER] markedly reduced mitochondrial membrane potential [MMP] in insulin-secreting INS cells, which was followed by a significant accumulation of cytochrome c [Cyt c] into the cytosolic compartment. In a manner akin to CER, exposure of these cells to interleukin-1beta [IL-1beta] also resulted in reduction in MMP and cytosolic accumulation of Cyt c. Further, long-term exposure of these cells to either CER [but not its inactive analogue] or IL-1beta caused a marked reduction in their metabolic viability. However, unlike IL-1beta, which increased nitric oxide [NO] release, CER-treatment of INS cells had no effects of CER on NO release were demonstrable. Together, these findings suggest that CER-induced mitochondrial effects may not be mediated via iNOS gene expression and NO production. CER also activated an okadaic acid -sensitive protein phosphatase [CAPP] in the purified mitochondrial fraction, suggesting that CAPP might represent one of the target proteins for CER in the beta cell mitochondria. Together, our findings suggest direct detrimental effects of CER on mitochondrial function in beta cells leading to their dysfunction and demise via apoptosis. Moreover, our findings provide evidence for a potential difference in the mechanisms underlying CER- and IL-1beta-induced mitochondrial defects and apoptotic demise of the effete beta cell.